The transcriptome of the rat subfornical organ is altered in response to early postnatal overnutrition.

Early postnatal overnutrition in humans is associated with long-term negative outcomes including obesity, increased risk of type-II diabetes, and cardiovascular disease. Hypothalamic neurons from rodents exposed to early postnatal overnutrition show altered expression of satiety signals and receptors, and exhibit altered responses to many satiety signals, suggesting a hypothalamic link between early overnutrition and development of these sequelae. Importantly, several hypothalamic nuclei receive information regarding circulating hormones (such as insulin, leptin and ghrelin) from the subfornical organ (SFO), a forebrain sensory circumventricular organ which lacks a blood brain barrier. Previous transcriptomic studies indicate that challenges to energy balance and hydration status stimulate changes in gene expression within the SFO, including genes encoding ion channels and receptors. In order to determine if early postnatal overnutrition also causes changes in SFO gene expression which may be associated with homeostatic dysregulation, we performed whole transcriptome sequencing on SFO tissue from rats raised in small (4 pups), or control (large, 12 pups) litters. Illumina RNA sequencing was performed on SFO tissue from rats raised from small and large litters, and read sequences were aligned to the Rat Rnor_6.0 genome. Control data were further compared to previously published microarray data set for validation. We found statistically significant (p < 0.05) changes in expression of 12 transcripts, three of which have likely roles in neuronal excitability, neurite outgrowth and differentiation, and food intake (Manf, Slc24a4, Cracr2b). Additionally, gene ontology analysis identified a trend among significantly altered transcripts in roles for oxidative stress response. We conclude that the SFO transcriptome is subtly altered by early postnatal overnutrition, and recommend further investigation of the effect of early postnatal overnutrition on SFO physiology and morphology.

The subfornical organ: A novel site for prolactin action.

Prolactin (PRL) is a peptide hormone that performs over 300 biological functions, including those that require binding to prolactin receptor (PRL-R) in neurones within the central nervous system (CNS). To enter the CNS, circulating PRL must overcome the blood-brain barrier. Accordingly, areas of the brain that do not possess a blood-brain barrier, such as the subfornical organ (SFO), are optimally positioned to interact with systemic PRL. The SFO has been classically implicated in energy and fluid homeostasis but has the potential to influence oestrous cyclicity and gonadotrophin release, which are also functions of PRL. We aimed to confirm and characterise the expression of PRL-R in the SFO, as well as identify the effects of PRL application on membrane excitability of dissociated SFO neurones. Using a quantitative real-time polymerase chain reaction, we found that PRL-R mRNA in the SFO of male and female Sprague Dawley rats did not significantly differ between juvenile and sexually mature rats (P = .34), male and female rats (P = .97) or across the oestrous cycle (P = .54). Patch-clamp recordings were obtained in juvenile male rats to further investigate the actions of PRL at the SFO. Dissociated SFO neurones perfused with 1 µmol L-1 PRL resulted in 2 responsive subpopulations of neurones; 40% depolarised (n = 15/43, 11.3 ± 1.7 mV) and 14% hyperpolarised (n = 6/43, -6.7 ± 1.4 mV) to PRL application. Within the range of 10 pmol L-1 to 1 µmol L-1 , the concentrations of PRL were not significantly different in either the magnitude (P = .53) or proportion (P = .19) of response. Furthermore, PRL application significantly reduced the transient K+ current in 67% of SFO neurones in voltage-clamp configuration (n = 6/9, P = .02). The stability in response to PRL and expression of PRL-R in the SFO suggests that PRL function is conserved across physiological states and circulating PRL concentrations, prompting further investigations aiming to clarify the nature of PRL function in the SFO.

Glucose concentrations modulate brain-derived neurotrophic factor responsiveness of neurones in the paraventricular nucleus of the hypothalamus.

The hypothalamic paraventricular nucleus (PVN) is critical for normal energy balance and has been shown to contain high levels of both brain-derived neurotrophic factor (BDNF) and tropomyosin-receptor kinase B mRNA. Microinjections of BDNF into the PVN increase energy expenditure, suggesting that BDNF plays an important role in energy homeostasis through direct actions in this nucleus. The present study aimed to examine the postsynaptic effects of BDNF on the membrane potential of PVN neurones, and also to determine whether extracellular glucose concentrations modulated these effects. We used hypothalamic PVN slices from male Sprague-Dawley rats to perform whole cell current-clamp recordings from PVN neurones. BDNF was bath applied at a concentration of 2 nmol L-1 and the effects on membrane potential determined. BDNF caused depolarisations in 54% of neurones (n=25; mean±SEM, 8.9±1.2 mV) and hyperpolarisations in 23% (n=11; -6.7±1.4 mV), whereas the remaining cells were unaffected. These effects were maintained in the presence of tetrodotoxin (n=9; 56% depolarised, 22% hyperpolarised, 22% nonresponders), or the GABAa antagonist bicuculline (n=12; 42% depolarised, 17% hyperpolarised, 41% nonresponders), supporting the conclusion that these effects on membrane potential were postsynaptic. Current-clamp recordings from PVN neurones next examined the effects of BDNF on these neurones at varying extracellular glucose concentrations. Larger proportions of PVN neurones hyperpolarised in response to BDNF as the glucose concentrations decreased [10 mmol L-1 glucose 23% (n=11) of neurones hyperpolarised, whereas, at 0.2 mmol L-1 glucose, 71% showed hyperpolarising effects (n=12)]. Our findings reveal that BDNF has direct GABAA independent effects on PVN neurones, which are modulated by local glucose concentrations. The latter observation further emphasises the critical importance of using physiologically relevant conditions in an investigation of the central pathways involved in the regulation of energy homeostasis.

Cellular actions of nesfatin-1 in the subfornical organ.

Nesfatin-1, a centrally acting anorexigenic peptide, is produced in several brain areas involved in metabolic processes and has been implicated in the control of ingestive behaviours and cardiovascular functions. The present study aimed to determine whether the subfornical organ (SFO), a central nervous system (CNS) site that has been extensively implicated in the regulation of appetite and thirst, may represent a potential site for central actions of nesfatin-1. We first used the reverse transcriptase-polymerase chain reaction and were able to confirm the presence of mRNA for the nucleobindin-2 gene in the SFO. We then used whole-cell patch clamp recordings to investigate the influence of nesfatin-1 on the membrane potential of dissociated SFO neurones. A total of 80.3% (49 of 61) of neurones tested showed a response to nesfatin-1 (100 nm, 10 nm and 1 nm). Of these, 47.5% depolarised, with a mean depolarisation of 8.2 ± 0.9 mV (n = 29) and 32.8% hyperpolarised with a mean hyperpolarisation of -8.9 ± 1.2 mV (n = 20). Peak magnitudes were seen at a concentration of 1 nm nesfatin-1, whereas no effect was observed at 100 pm nesftain-1 (n = 3). Furthermore, voltage clamp ramp and step protocols revealed a nesfatin-1 induced activation of the delayed rectifier potassium conductance, IK . Pharmacological blockade of this conductance greatly reduced the magnitude and occurrence of the observed hyperpolarisations. The present study thus demonstrates that nesfatin-1 has the ability to influence the membrane potential of SFO neurones, and thus identifies the SFO as a potential site at which nesfatin-1 may act to regulate ingestive behaviour and cardiovascular control.

α-MSH exerts direct postsynaptic excitatory effects on NTS neurons and enhances GABAergic signaling in the NTS.

The central melanocortin system plays an essential role in the regulation of energy balance. While anorexigenic effects of α-melanocyte-stimulating hormone (α-MSH) acting in the nucleus of the solitary tract (NTS), a critical medullary autonomic control center, have been established, the cellular events underlying these effects are less well characterized. In this study, we used whole-cell patch-clamp electrophysiology to examine firstly whether α-MSH exerts direct postsynaptic effects on the membrane potential of rat NTS neurons in slice preparation, and secondly whether α-MSH influences GABAergic signaling in the NTS. In normal artificial cerebrospinal fluid, perfusion of α-MSH (500 nM) resulted in a depolarization in 39% of cells (n=16, mean 6.14±0.54 mV), and a hyperpolarization in 22% of cells (n=9, -6.79±1.02 mV). Studies using tetrodotoxin to block neuronal communication revealed α-MSH exerts direct depolarizing effects on some NTS neurons, and indirect inhibitory effects on others. A third subset of neurons is simultaneously directly depolarized and indirectly hyperpolarized by α-MSH, resulting in a net lack of effect on membrane potential. The inhibitory inputs influenced by α-MSH were identified as GABAergic, as α-MSH increased the frequency, but not amplitude, of inhibitory postsynaptic currents (IPSCs) in 50% of NTS neurons. α-MSH had no effect on the frequency or amplitude of miniature IPSCs. Furthermore, pharmacological blockade of GABAA and GABAB receptors, and physical removal of all synaptic inputs via cellular dissociation, abolished hyperpolarizations induced by α-MSH. We conclude α-MSH exerts direct, postsynaptic excitatory effects on a subset of NTS neurons. By exciting GABAergic NTS neurons and presynaptically enhancing GABAergic signaling, α-MSH also indirectly inhibits other NTS cells. These findings provide critical insight into the cellular events underlying medullary melanocortin anorexigenic effects, and expand the understanding of the circuitries involved in central melanocortin signaling.

Glucose-responsive neurons in the subfornical organ of the rat--a novel site for direct CNS monitoring of circulating glucose.

Glucose-sensitive neurons have been identified in a number of CNS regions including metabolic control centers of the hypothalamus. The location of these regions behind the blood-brain barrier restricts them to sensing central, but not circulating glucose concentrations. In this study, we have used patch-clamp electrophysiology to examine whether neurons in a specialized region lacking the blood-brain barrier, the subfornical organ (SFO), are also glucose sensitive. In dissociated SFO neurons, altering the bath concentration of glucose (1 mM, 5 mM, 10 mM) influenced the excitability of 49% of neurons tested (n=67). Glucose-inhibited (GI) neurons depolarized in response to decreased glucose (n=10; mean, 4.6±1.0 mV) or hyperpolarized in response to increased glucose (n=8; mean,-4.4±0.8 mV). In contrast, glucose-excited (GE) neurons depolarized in response to increased glucose (n=9; mean, 6.4±0.4 mV) or hyperpolarized in response to decreased glucose (n=6; mean,-4.8±0.6 mV). Using voltage-clamp recordings, we also identified GI (outward current to increased glucose) and GE (inward current to increased glucose) SFO neurons. The mean glucose-induced inward current had a reversal potential of -24±12 mV (n=5), while GE responses were maintained during sodium-dependent glucose transporter inhibition, supporting the conclusion that GE properties result from the activation of a nonselective cation conductance (NSCC). The glucose-induced outward current had a mean reversal potential of -78±1.2 mV (n=5), while GI responses were not observed in the presence of glibenclamide, suggesting that these properties result from the modulation of K(ATP) channels. These data demonstrate that SFO neurons are glucose responsive, further emphasizing the potential roles of this circumventricular organ as an important sensor and integrator of circulating signals of energy status.

Cardiovascular actions of leptin in the subfornical organ are abolished by diet-induced obesity.

The subfornical organ (SFO), a sensory circumventricular organ lacking the normal blood-brain barrier with well documented roles in cardiovascular regulation, has recently been identified as a potential site at which the adipokine, leptin, may act to influence central autonomic pathways. Systemic and central leptin administration has been shown to increase blood pressure and it has been suggested that selective leptin resistance contributes to obesity-related hypertension. Given the relationship between obesity and hypertension, the present study aimed to investigate the cardiovascular consequences of the direct administration of leptin into the SFO of young lean rats and in the diet-induced obesity (DIO) rat model, which has been shown to be leptin-resistant. Leptin administration (500 fmol) directly into the SFO of young rats resulted in rapid decreases in blood pressure (BP) [mean area under the curve (AUC) = -677.8 ± 167.1 mmHg*s; n = 9], without an effect on heart rate (mean AUC = -21.2 ± 13.4 beats; n = 9), and these effects were found to be dose-related as microinjection of 5 pmol of leptin into the SFO had a larger effect on BP (mean AUC = -972.3 ± 280.1 mmHg*s; n = 4). These BP effects were also shown to be site-specific as microinjection of leptin into non-SFO regions or into the ventricle was without effect on BP (non-SFO: mean AUC = -22.4 ± 55.3 mmHg*s; n = 4; ventricle: mean AUC = 194.0 ± 173.0 mmHg*s; n = 6). By contrast, microinjection of leptin into leptin-resistant DIO rats was without effect on BP (mean AUC = 205.2 ± 75.1 mmHg*s; n = 4). These observations suggest that the SFO may be an important relay centre through which leptin, in normal weight, leptin responsive rats, acts to maintain BP within normal physiological limits through descending autonomic pathways involved in cardiovascular control and that, in obese, leptin-resistant, rats leptin no longer influences SFO neurones, resulting in an elevated BP, thus contributing to obesity-related hypertension.

Orexin receptor subtype activation and locomotor behaviour in the rat.

AIM: Orexin-producing neurones, located primarily in the perifornical region of the lateral hypothalamus, project to a wide spectrum of brain sites where they influence numerous behaviours as well as modulating the neuroendocrine and autonomic responses to stress. While some of the actions of orexin appear to be mediated via the type 1 receptor, some are not, including its action on the release of one stress hormone, prolactin. We describe here the ability of orexin to increase locomotor behaviours and identify the importance of both receptor subtypes in these actions. METHODS: Rats were tested for their behavioural responses to the central activation of both the type 1 (OX(1)R) and type 2 (OX(2)R) receptor (ICV orexin A), compared to OX(2)R activation using a relatively selective OX(2)R agonist in the absence or presence of an orexin receptor antagonist that possesses highest affinity for OX(1)R. RESULTS: Increases in locomotor activity were observed, effects which were expressed by not only orexin A, which binds to both the OX(1)R and the OX(2)R receptors, but also by the relatively selective OX(2)R agonist [(Ala(11), Leu(15))-orexin B]. Furthermore, the OX(1)R selective antagonist only partially blocked the action of orexin A on most locomotor behaviours and did not block the actions of [(Ala(11), Leu(15))-orexin B]. CONCLUSION: We conclude that orexin A exerts its effects on locomotor behaviour via both the OX(1)R and OX(2)R and that agonism or antagonism of only one of these receptors for therapeutic purposes (i.e. sleep disorders) would not provide selectivity in terms of associated behavioural side effects.

Neuropeptide W has cell phenotype-specific effects on the excitability of different subpopulations of paraventricular nucleus neurones.

The administration of the neuropeptide W (NPW) and neuropeptide B (NPB) in rodents has been shown to influence the activity of a variety of autonomic and neuroendocrine systems. The paraventricular nucleus (PVN) is a major autonomic and neuroendocrine integration site in the hypothalamus, and neurones within this nucleus express the receptor for these ligands, NPB/W receptor 1 (NPBWR1). In the present study, we used whole cell patch clamp recordings coupled with single-cell reverse transcriptase-polymerase chain reaction to examine the effects of neuropeptide W-23 (NPW-23) on the excitability of identified PVN neurones. Oxytocin, vasopressin and thyrotrophin-releasing hormone neurones were all found to be responsive to 10 nm NPW-23, although both depolarising and hyperpolarising effects were observed in each of these cell groups. By contrast, corticotrophin-releasing hormone cells were unaffected. Further subdivision of chemically phenotyped cell groups into magnocellular, neuroendocrine or pre-autonomic neurones, using their electrophysiological fingerprints, revealed that neurones projecting to medullary and spinal targets were predominantly inhibited by NPW-23, whereas those that projected to median eminence or neural lobe showed almost equivalent numbers of depolarising and hyperpolarising cells. The demonstration of particular phenotypic populations of PVN neurones showing NPW-induced effects on excitability reinforces the importance of the NPB/NPW neuropeptide system as a regulator of autonomic function.

Gastrointestinal hormone actions in the central regulation of energy metabolism: potential sensory roles for the circumventricular organs.

A variety of circulating signals provide essential information to the central nervous system (CNS) regarding nutritional status. The gastrointestinal system produces many such molecules that are now known to have profound effects on feeding behavior and the control of metabolism as a consequence of their ability to regulate the neural circuitry involved in metabolic homeostasis. Although many of these substances have been suggested to directly access such brain centers, their lipophobic characteristics suggest that alternative mechanisms should be considered. In this paper, we consider one such alternative, namely, that a specialized group of CNS structures collectively known as the sensory circumventricular organs (CVOs), which are not protected by the normal blood-brain barrier, may play important roles in such blood to brain communications. Specifically, we review a developing literature that shows receptors for, and functional actions of, gastrointestinal hormones such as amylin, cholecystokinin, ghrelin and peptide YY in the area postrema and subfornical organ. Collectively, these observations suggest potentially significant roles for the sensory CVOs in the regulation of energy balance.

The subfornical organ: a central nervous system site for actions of circulating leptin.

Adipose tissue plays a critical role in energy homeostasis, secreting adipokines that control feeding, thermogenesis, and neuroendocrine function. Leptin is the prototypic adipokine that acts centrally to signal long-term energy balance. While hypothalamic and brain stem nuclei are well-established sites of action of leptin, we tested the hypothesis that leptin signaling occurs in the subfornical organ (SFO). The SFO is a circumventricular organ (CVO) that lacks the normal blood-brain barrier, is an important site in central autonomic regulation, and has been suggested to have a role in modulating peripheral signals indicating energy status. We report here the presence of mRNA for the signaling form of the leptin receptor in SFO and leptin receptor localization by immunohistochemistry within this CVO. Central administration of leptin resulted in phosphorylation of STAT3 in neurons of SFO. Whole cell current-clamp recordings from dissociated SFO neurons demonstrated that leptin (10 nM) influenced the excitability of 64% (46/72) of SFO neurons. Leptin was found to depolarize the majority of responsive neurons with a mean change in membrane potential of 7.3 +/- 0.6 mV (39% of all SFO neurons), while the remaining cells that responded to leptin hyperpolarized (-6.9 +/- 0.7 mV, 25% of all SFO neurons). Similar depolarizing and hyperpolarizing effects of leptin were observed in recordings from acutely prepared SFO slice preparations. Leptin was found to influence the same population of SFO neurons influenced by amylin as three of four cells tested for the effects of bath application of both amylin and leptin depolarized to both peptides. These observations identify the SFO as a possible central nervous system location, with direct access to the peripheral circulation, at which leptin may act to influence hypothalamic control of energy homeostasis.

Nesfatin-1 influences the excitability of paraventricular nucleus neurones.

Nesfatin-1 is a newly-discovered satiety peptide found in several nuclei of the hypothalamus, including the paraventricular nucleus. To begin to understand the physiological mechanisms underlying these satiety-inducing actions, we examined the effects of nesfatin-1 on the excitability of neurones in the paraventricular nucleus. Whole-cell current-clamp recordings from rat paraventricular nucleus neurones showed nesfatin-1 to have either hyperpolarizing or depolarising effects on the majority of neurones tested. Both types of response were observed in neurones irrespective of classification based on electrophysiological fingerprint (magnocellular, neuroendocrine or pre-autonomic) or molecular phenotype (vasopressin, oxytocin, corticotrophin-releasing hormone, thyrotrophin-releasing hormone or vesicular glutamate transporter), determined using single cell reverse transcription-polymerase chain reaction. Consequently, we provide the first evidence that this peptide, which is produced in the paraventricular nucleus, has effects on the membrane potential of a large proportion of different subpopulations of neurones located in this nucleus, and therefore identify nesfatin-1 as a potentially important regulator of paraventricular nucleus output.

Cardiovascular actions of orexin-A in the rat subfornical organ.

Orexin-A is a neuropeptide, primarily produced in the lateral hypothalamic/perifornical hypothalamus. Orexin receptors and immunoreactive neuronal fibres are widely distributed throughout the brain, suggesting integrative neurotransmitter roles in a variety of physiological systems. Intracerebroventricular injections of orexin-A increase blood pressure and stimulate drinking, and the subfornical organ (SFO), a circumventricular structure implicated in autonomic control, is a potential site at which orexin may act to exert these effects. We have therefore used microinjection techniques to examine the effects of orexin-A administered directly into the SFO on blood pressure and heart rate in urethane anaesthetised male Sprague-Dawley rats. Orexin-A microinjection (50 fmol) into the SFO caused site-specific decreases in blood pressure (SFO: mean area under curve (AUC) = -681.7 +/- 46.8 mmHg*s, n = 22 versus non-SFO: 63.68 +/- 54.69 mmHg*s, n = 15, P < 0.001), and heart rate (SFO: mean AUC = -26.7 +/- 2.8 beats, n = 22, versus non-SFO: mean AUC = 1.62 +/- 2.1 beats, n = 15, P < 0.001). Vagotomy did not alter the hypotensive or bradycardic responses elicited by orexin-A microinjection. Prior alpha-adrenoceptor blockade with phenoxybenzamine (1 mg/kg, i.v.) masked the orexin-A induced blood pressure (mean AUC = -122.6 +/- 17.6 mmHg*s, n = 4, P < 0.01 paired t-test) and heart rate (mean AUC = -6.7 +/- 1.7 beats, n = 4, P < 0.05, paired test) response. The orexin-A induced heart rate response was attenuated when beta-adrenoceptors were blocked with propranolol (1 mg/kg, i.v.; mean AUC = 0.6 +/- 2.8 beats, n = 5, P < 0.01 paired t-test). These studies demonstrate that microinjection of orexin-A into the SFO causes site specific decreases in blood pressure and heart rate which is mediated by a reduction in sympathetic tone.

Prostaglandin E2 mediates cellular effects of interleukin-1beta on parvocellular neurones in the paraventricular nucleus of the hypothalamus.

Abstract Interleukin-1beta (IL-1beta) is involved in hypothalamic regulation of corticotrophin-releasing hormone secretion, autonomic activation and consequent downstream modulation of the neuroimmune response. Previously, we have shown that IL-1beta depolarises parvocellular neurones in the paraventricular nucleus (PVN) of the hypothalamus, and these effects are dependent on attenuation of gamma-amino butyric acid (GABA)-ergic input. In the present study, using whole-cell patch clamp recordings of rat neurones in a slice preparation of the PVN, we show that the effects of IL-1beta are abolished in the presence of a cyclooxygenase (COX)-2 inhibitor, NS-398, indicating a dependence on prostaglandin (PG) synthesis and activation. In response to 1 microM PGE2, 64% of parvocellular neurones tested exhibited a clear depolarisation, which was abolished in the presence of tetrodotoxin (TTX). Furthermore, neurones responsive to both IL-1beta and PGE2 exhibited a decrease in the frequency of inhibitory post-synaptic potentials, suggesting that effects of these modulators are mediated via a decrease in GABA-ergic input to these neurones. A proportion (44% and 40%, respectively) of putative GABA-ergic neurones in the halo region surrounding the PVN demonstrated hyperpolarising responses to 1 nM IL-1beta and 1 microM PGE2, and these effects were maintained in TTX. Furthermore, direct hyperpolarising effects of IL-1beta were blocked in the presence of NS-398. Together, these data suggest that PGE2, synthesised in response to IL-1beta-activation of COX-2 expressing cells, directly hyperpolarises putative GABA-ergic neurones in the halo zone surrounding and projecting to the PVN, resulting in a decrease in GABA-ergic input to parvocellular neurones and consequent depolarisation. These data further elucidate the cellular mechanisms by which IL-1beta exerts its neuroimmune-related actions in the PVN.

ANG II-induced excitation of paraventricular nucleus magnocellular neurons: a role for glutamate interneurons.

The hypothalamic paraventricular nucleus (PVN) plays a critical role in cardiovascular and neuroendocrine regulation. ANG II (ANG) acts throughout the periphery in the maintenance of fluid-electrolyte homeostasis and has also been demonstrated to act as a neurotransmitter in PVN exerting considerable influence on neuronal excitability in this nucleus. The mechanisms underlying the ANG-mediated excitation of PVN magnocellular neurons have yet to be determined. We have used whole cell patch-clamp techniques in hypothalamic slices to examine the effects of ANG on magnocellular neurons. Application of ANG resulted in a depolarization of magnocellular neurons, a response that was abolished in TTX, suggesting an indirect mechanism of action. Interestingly, ANG also increased the frequency of excitatory postsynaptic potentials/currents in magnocellular neurons, an effect that was abolished after application of the glutamate antagonist kynurenic acid. ANG was without effect on the amplitude of excitatory postsynaptic currents, suggesting a presynaptic action on an excitatory interneuron within PVN. The ANG-induced depolarization was shown to be sensitive to kynurenic acid, revealing the requisite role of glutamate in mediating the ANG-induced excitation of magnocellular neurons. These observations indicate that the ANGergic excitation of magnocellular PVN neurons are dependent on an increase in glutamatergic input and thus highlight the importance of a glutamate interneuron in mediating the effects of this neurotransmitter.

Interleukin-1 beta depolarizes paraventricular nucleus parvocellular neurones.

Interleukin-1 beta (IL-1 beta) is involved in hypothalamic regulation of corticotropin releasing hormone (CRH) secretion and consequent downstream modulation of the neuroimmune response. In this study, whole-cell patch clamp recordings of rat parvocellular neurones in a slice preparation of the paraventricular nucleus (PVN) of the hypothalamus were performed to examine the cellular effects of IL-1 beta. In response to 1 nm IL-1 beta, 65% of parvocellular neurones tested exhibited a clear depolarization, which was abolished in the presence of tetrodotoxin (TTX). This depolarization was partially dependent on nitric oxide formation, as demonstrated by attenuation of the response in the presence of N-omega-nitro-L-arginine methylester, a nitric oxide synthase inhibitor. The effects of IL-1 beta on responsive parvocellular neurones were associated with a decrease in the frequency of inhibitory post synaptic potentials (IPSPs). Bicuculline administration blocked the effects of IL-1 beta, suggesting that this cytokine modulates GABA-ergic output, resulting in a decrease in inhibitory input (IPSPs) and consequent depolarization. These data support the conclusion that IL-1 beta influences the excitability of parvocellular neurones in the PVN, as a secondary consequence of nitric oxide generation and modulation of GABAergic inhibitory input to these cells. They elucidate cellular correlates underlying the well-established neuroimmune roles of IL-1 beta in the paraventricular nucleus of the hypothalamus.

Selective potentiation of N-type calcium channels by angiotensin II in rat subfornical organ neurones.

1. Here we have characterized Ca(2+) currents in rat subfornical organ neurones, and their modulation by angiotensin II. Currents were of the high voltage-activated (HNA) subtype, as the threshold for activation was near -30 mV (mid-point potential (V(50)) of activation -14 mV). Using Ba(2+) as the charge carrier, little inactivation was observed, and it occurred only at depolarized potentials (V(50) of inactivation -12 mV). More inactivation was observed using Ca(2+) as the charge carrier, indicating that Ca(2+)-dependent inactivation plays a role in regulating Ca(2+) channel function in subfornical organ (SFO) neurones. 2. The net Ba(2+) current could be blocked by Cd(2+) (EC(50) 1.6 microM), confirming that currents are of the HVA variety. By using selective antagonists, we identified the presence of both L- and N-type channels; 20 microM nifedipine blocked 22 +/- 1 % of the current, while omega-conotoxin GVIA blocked 65 +/- 7 %, indicating that these currents make up the net current through Ca(2+) channels. 3. Angiotensin II potentiated the inward current throughout the range of activation. Using depolarizing voltage ramps, 1 nM angiotensin potentiated the peak current by 14 +/- 5 %. We then used selective blockade of the HVA component currents; 20 microM nifedipine failed to prevent the potentiation by angiotensin II (12 +/- 5 %), while blocking N-type channels with omega-conotoxin GVIA blocked the facilitation by ANG (2.3 +/- 2 %). Losartan (1 microM) prevented the actions of ANG on the inward current (1.6 +/- 1 %), indicating that the selective effects of ANG on N-type channels in SFO neurones are mediated by AT(1) receptors.

Slowly inactivating potassium conductance (I(D)): a potential target for stroke therapy.

BACKGROUND AND PURPOSE: Excessive accumulation of extracellular glutamate results in the death of most, but not all, neurons in the central nervous system. Understanding the unique properties of cells that can withstand this excitotoxic challenge may identify specific targets for novel stroke therapies. METHODS: A combination of in vivo methods for analysis of excitotoxic cell death after activation of N-methyl-D-aspartate (NMDA) receptors and in vitro patch-clamp analysis of specific conductances in hypothalamic slices and dissociated cells has been used to assess the roles of specific potassium conductances in delayed cell death after NMDA receptor activation. RESULTS: We report that a specific D-type potassium conductance (I(D)), necessary for the rapid repolarization of the membrane after a strong depolarization, serves such a protective purpose in magnocellular neurons of the paraventricular nucleus. Manipulations that inhibit this current (4-aminopyridine or angiotensin II) increase neuronal excitability and augment cell death after NMDA receptor activation. In addition, this protection is not observed in magnocellular neurons of spontaneously hypertensive rats, and intriguingly it can be reestablished by blocking angiotensin II receptors in these animals. CONCLUSIONS: These observations provide a persuasive experimental explanation for the unexpected finding that therapeutic treatments for hypertension that block central as well as peripheral angiotensin type 1 receptors reduce the severity and occurrence of stroke.

Subfornical organ neurons projecting to paraventricular nucleus: whole-cell properties.

The subfornical organ (SFO) has been repeatedly identified as a CNS site that plays a critical role in sensing multiple physiological variables of the "milieu interieur" and, through efferent projections to other CNS sites, initiating physiological responses to change. Many recent in vitro patch-clamp studies have examined the cellular mechanisms underlying the sensory abilities of these specialized CNS neurons. The primary limitation of these studies, however, has been the inability to identify homogeneous groups of SFO neurons for such investigation. We report here the development of techniques to permit patch clamp recording from dissociated SFO neurons identified according to their in vivo projection site. SFO neurons were labeled by injection of fluorescently labeled, retrogradely transported microspheres into the hypothalamic paraventricular nucleus (PVN) 3 days prior to cell dissociation. Patch-clamp recordings from these SFO-PVN neurons revealed both sodium currents, potassium currents, action potentials, input resistance and membrane potential which were all similar to SFO cells prepared from animals with no prior tracer injection. Labeled SFO-->PVN cells were also found to be osmosensitive and responsive to angiotensin II, suggesting specific functional roles for this anatomically defined group of SFO neurons. Intriguingly, our post hoc analysis also demonstrated that all labeled neurons demonstrated a unique electrophysiological profile dominated by a large transient potassium conductance such that the transient/sustained potassium current ratio, or degree of inactivation was, on average, greater than 4.0. Utilization of these tracing techniques to permit the in vitro recording from cells with known in vivo connections will permit study of intrinsic mechanisms that underlie physiological responses of anatomically defined populations of neurons.

Hormonal and neurotransmitter roles for angiotensin in the regulation of central autonomic function.

In this review we present the case for both hormonal and neurotransmitter actions of angiotensin II (ANG) in the control of neuronal excitability in a simple neural pathway involved in central autonomic regulation. We will present both single-cell and whole-animal data highlighting hormonal roles for ANG in controlling the excitability of subfornical organ (SFO) neurons. More controversially we will also present the case for a neurotransmitter role for ANG in SFO neurons in controlling the excitability of identified neurons in the paraventricular nucleus (PVN) of the hypothalamus. In this review we highlight the similarities between the actions of ANG on these two populations of neurons in an attempt to emphasize that whether we call such actions "hormonal" or "neurotransmitter" is largely semantic. In fact such definitions only refer to the method of delivery of the chemical messenger, in this case ANG, to its cellular site of action, in this case the AT1 receptor. We also described in this review some novel concepts that may underlie synthesis, metabolic processing, and co-transmitter actions of ANG in this pathway. We hope that such suggestions may lead ultimately to the development of broader guiding principles to enhance our understanding of the multiplicity of physiological uses for single chemical messengers.